dynamics and energetics of solute permeation through the ... · dynamics and energetics of solute...

Dynamics and energetics of solute permeation through the

Plasmodium falciparum aquaglyceroporin

Camilo Aponte-Santamarıa,a Jochen S. Hubb and Bert L. de Groot*a

Received 16th March 2010, Accepted 15th June 2010

DOI: 10.1039/c004384m

The aquaglyceroporin from Plasmodium falciparum (PfAQP) is a potential drug target for the

treatment of malaria. It efficiently conducts water and other small solutes, and is proposed to

intervene in several crucial physiological processes during the parasitic life cycle. Despite the

wealth of experimental data available, a dynamical and energetic description at the single-

molecule level of the solute permeation through PfAQP has been lacking so far. Here we address

this question by using equilibrium and umbrella sampling molecular dynamics simulations.

We computed the water osmotic permeability coefficient, the pore geometry and the potential of

mean force for the permeation of water, glycerol and urea. Our simulations show that the PfAQP,

the human aquaporin 1 (hAQP1) and the Escherichia coli glycerol facilitator (GlpF) have nearly

identical water permeabilities. The Arg196 residue at the ar/R region was found to play a crucial

role regulating the permeation of water, glycerol and urea. The computed free energy barriers at

the ar/R selectivity filter corroborate that PfAQP conducts glycerol at higher rates than urea, and

suggest that PfAQP is a more efficient glycerol and urea channel than GlpF. Our results are

consistent with a solute permeation mechanism for PfAQP which is similar to the one established

for other members of the aquaglyceroporin family. In this mechanism, hydrophobic regions near

the NPA motifs are the main water rate limiting barriers, and the replacement of water–arg196

interactions and solute-matching in the hydrophobic pocket at the ar/R region are the main

determinants underlying selectivity for the permeation of solutes like glycerol and urea.

1. Introduction

The parasite Plasmodium falciparum is responsible for the

most lethal form of the malaria disease.1 It expresses one

aquaglyceroporin (PfAQP),2 that has been proposed to play

crucial physiological roles during the parasitic life cycle,2,3 and

is therefore a potential antimalarial drug target.4–6

PfAQP has been intensively studied experimentally over the

last decade. Functional studies2 established that PfAQP con-

ducts water and glycerol at high permeability rates, comparable

to the rates of the human aquaporin 1 (hAQP1) in the case

of water. These experiments, therefore, suggested that PfAQP

is implicated in tasks such as osmotic stress regulation, and

glycerol uptake for lipid synthesis and oxidative stress regula-

tion.2 In addition, PfAQP was found to be permeable to urea,2

ammonia,7 other polyols up to five carbons long,2 carbonyl

compounds3 and arsenite,2 suggesting that PfAQP may also be

involved in the release of toxic products. The biological role of

aquaglyceroporins for the Plasmodium parasites was further

investigated in experiments deleting the orthologue of PfAQP

in the rodent malaria parasite, Plasmodium berghei (PbAQP),

that showed a slower proliferation of the parasite in infected

mice.8 Furthermore, mutation studies demonstrated that a

glutamate residue located at the C loop (Glu125) near the

conserved Arg196 is critical for water permeation.9

The structure of PfAQP was recently determined by X-ray

crystallography at 2.05 A resolution.10 It revealed the 3D

architecture of the channel and provided molecular insights

into the efficient water and glycerol permeation. PfAQP has

the same fold as compared to other members of the family of

aquaglyceroporins.11 The pore geometry is remarkably similar

to the Escherichia coli glycerol facilitator (GlpF),10 the closest

homologous aquaglyceroporin (with a known structure) with

a sequence similarity of 50%.2 PfAQP arranges in a tetrameric

structure, where each monomeric unit constitutes a conduc-

tion pore. The conduction pore contains two constriction

regions: one located near the NPA motifs, that are replaced

in PfAQP by unusual NLA and NPS motifs, and a second

one at the ar/R region, where Arg196 is located facing the

hydrophobic Trp50 and Phe190 residues.10 In addition, the C

loop is anchored to the extracellular vestibule with Glu125

located in proximity to Arg196, highlighting on the impor-

tance of this region for the solute permeation,10 as previously

hypothesized from functional studies.9

Despite the wealth of data available, a dynamical and

energetic description at the single-molecule level of solute

permeation through PfAQP is lacking so far. Furthermore, a

systematic comparison of PfAQP with hAQP1 and GlpF (the

most studied aquaporin and aquaglyceroporin, respectively),

in terms of the solute permeation and their dynamical causes,

is incomplete. The goal of this paper is to address these two

questions employing molecular dynamics simulations, based

on the X-ray crystallographic structure of PfAQP. Initially,

the water permeation through PfAQP was quantified by

a Computational Biomolecular Dynamics Group, Max Planck Institutefor Biophysical Chemistry, Gottingen, Germany

bComputational and Systems Biology, Dept. of Cell and MolecularBiology, Uppsala University, Uppsala, Sweden

10246 | Phys. Chem. Chem. Phys., 2010, 12, 10246–10254 This journal is �c the Owner Societies 2010

PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

computing the single molecule osmotic permeability coefficient.

Subsequently, the pore geometry and the energetics for water

transport through PfAQP were analyzed by computing the

radius and potential of mean force profiles from equilibrium

simulations. Finally the energetics of permeation of glycerol

and urea was studied by computing potentials of mean force

by using the technique of umbrella sampling simulations.

The paper is organized as follows: section 2.1 lists the

parameters and algorithms used for the equilibrium molecular

dynamics simulations; section 2.2 to 2.4 describes the methods

to derive the water permeability coefficients, the pore geometry

and potentials of mean force from equilibrium MD simula-

tions, and section 2.5 describes the umbrella sampling simula-

tions. The results are presented and discussed in sections 3 and

4. Finally, section 5 summarizes our conclusions.

2. Theory and methods

2.1 Equilibrium molecular dynamics simulations

Equilibrium molecular dynamics simulations were carried out

starting with the aquaporin tetramer in a fully solvated

Dipalmitoylphosphatidylcholine (DPPC) lipid bilayer (Fig. 1).

Three independent simulations of PfAQP aquaporin (labeled

from I to III) were carried out: a control simulation of the

wild-type form (I); a mutant where Glu125 (located at the C

loop) was mutated into serine (II), and a second mutant where

Arg196 (located at the pore face) was replaced by alanine (III).

The latter two simulations were performed to study the effect

of these two residues on the water permeation (Fig. 1(c)). In

addition, simulations of hAQP1 (IV) and GlpF (V) aqua-

porins were also carried out for comparison.

The simulation boxes contain the protein tetramer, 265

DPPC lipids (263 in simulation V) and around 21 500 SPC/E

water molecules.12 The PfAQP and GlpF structures were

taken from the Protein Data Bank (PDB ID codes 3C0210

and 1FX8,13 respectively). The starting structure of hAQP1

was modeled based on the X-ray structure of bovine AQP1

(PDB ID code 1J4N)14 by mutating differing residues by using

the WHAT IF modeling software.15 The tetramer was inserted

into the lipid bilayer by using the g_membed software.16

Crystallographic water molecules were kept in the structures

and ions were added to neutralize the simulation systems. The

amber99SB all-atom force field17 was used for the protein,

and lipid parameters were taken from Berger et al.18 The

simulations were carried out using the GROMACS simulation

software.19,20 Long-range electrostatic interactions were

calculated with the particle-mesh Ewald method.21,22 Short-

range repulsive and attractive interactions were described by a

Lennard-Jones potential, which was cut off at 1.0 nm. The

Settle algorithm23 was used to constrain bond lengths and

angles of water molecules and Lincs24 was used to constrain all

other bond lengths. The fastest angular degrees of freedom

involving hydrogen atoms were removed by using the virtual

interaction-sites algorithm,25 allowing a time step of 4 fs. The

temperature was kept constant by coupling the system to a

velocity rescaling thermostat26,27 at 300 K with a coupling

constant t = 0.5 ps. The pressure was kept constant by

coupling the system to a semiisotropic Parrinello-Rahman

barostat28 at 1 bar with a coupling constant of t = 5.0 ps.

All simulations were equilibrated for 4 ns before production.

During this equilibration time the coordinates of the protein

were harmonically restrained, with a harmonic force constant

of 1000 kJ mol�1/nm2. The simulation length of the produc-

tion runs was 100 ns discarding the first 10 ns as equilibration.

2.2 Water permeability calculations

The single-channel osmotic permeability, pf, was chosen to

quantify the water permeation through the simulated

Fig. 1 Molecular dynamics simulations of PfAQP. Side (a) and top

views (b) of the simulation box illustrating the tetramer (red, orange,

cyan and blue), fully embedded in a DPPC lipid bilayer (yellow

phosphorous atoms and green tails) and solvated by water (red,

white). (c) Conduction pore showing the helices HB and HE; the C

loop (cyan), and Glu125 and Arg196 (green). The blue lines indicate

the region considered to compute the water permeability coefficient pf.

This journal is �c the Owner Societies 2010 Phys. Chem. Chem. Phys., 2010, 12, 10246–10254 | 10247

aquaglyceroporins. A solute concentration difference, DCs,

between two media connected by a channel causes a net water

flux, j, through the channel, and the permeability coefficient,

pf, relates these two quantities:

j = �pfDCs. (1)

The pf was independently calculated for every monomer, based

on the collective diffusion model proposed by Zhu et al.29 A

collective variable n is defined as

dn ¼X

i

dzi=L; ð2Þ

where dzi are the displacements along the pore coordinate of

the water molecules inside the channel (labeled as i) during a

time dt, and L is the length of the channel. The variable n

obeys an Einstein relation,

hn2(t)i = 2Dnt, (3)

and the proportionality constant, 2Dn, is related with the

permeability coefficient:29

pf = uwDn, (4)

where uw C 30 A3 is the volume occupied by one water

molecule.

Water molecule displacements, dzi, were computed every

2 ps, within a cylindrical region (of length L = 2.05 nm and

radius r = 0.5 nm) centered at the pore axis and spanning

�0.7 nm down and 1.35 nm up from the NPA region

(Fig. 1(c)). hn2(t)i was obtained by averaging over 450 time

windows of 200 ps length each. Finally the pf was obtained

from the slope of the curve hn2(t)i versus time. An effective

pf value was obtained by averaging the values of the four

monomers and the error was estimated as the standard

error.

2.3 Pore dimensions

The geometry of the channels was monitored by computing

pore radius profiles with the HOLE software,30 averaging over

snapshots taken every 50 ps, yielding a standard error always

smaller than 0.4 A

2.4 Potential of mean force for water

The potential of mean force (PMF) for water was calculated to

identify rate limiting regions and relevant water–protein and

water–water interaction sites inside the pore. It was independently

calculated for every monomer by using the formula31

Gi(z) = �kBT lnhni(z)i, (5)

where kB is the Boltzmann constant, T is the simulation

temperature and hni(z)i is the average (over the whole trajectory)of the number of water molecules at the z position along the

ith pore. The number of water molecules is referred to a

cylinder (of radius 0.5 nm) that is aligned with the pore

coordinate (Fig. 1(c)). Therefore, the entropy at the bulk

regions is underestimated, and consequently, the bulk free

energy is also underestimated. To relate G(z) with the area of

one aquaporin monomer and account for such understima-

tions, a trapezoidal correction was applied in the entrance and

exit regions. In consequence, the final PMF refers to a density

of one channel per membrane area occupied by an aquaporin

monomer.32 The correction reads DGcyl = kBT ln(Amono/Ac),

where Amono denotes the membrane cross section area of the

aquaporin monomer and Ac the cross section area of the

cylinder (p � 0.25 nm2). The computed correction was

6.1 kJ/mol for PfAQP, 6.0 kJ/mol for hAQP1 and 6.2 kJ/mol

for GlpF. Prior to the analysis the protein monomers were

superimposed to a reference structure. The effective PMF,

Geff(z), was computed by combining the four monomer Gi(z) as

expð�GeffðzÞ=kBTÞ ¼1

4

X4

i¼1expð�GiðzÞ=kBTÞ

¼ 1

4

X4

i¼1hniðzÞi;

ð6Þ

and the error of Geff was estimated by propagating the standard

errors of hni(z)i, yielding a maximum uncertainty of approxi-

mately 1.5 kJ/mol.

2.5 Umbrella sampling simulations

The starting frames for the umbrella simulation were taken

from a 10 ns equilibrium simulations of PfAQP. The simula-

tion system contained the PfAQP tetramer, 264 POPE lipids,

20484 TIP4P water molecules,33 and four chloride ions.

Protein and glycerol interactions were described using the

OPLS all-atom force field,34,35 lipid parameters were taken

from from Berger et al.,18 and urea parameters from ref. 36

and 37. All simulation parameters were chosen as described in

the section 2.1, except that hydrogen atoms were not described

as virtual sites, requiring a time step of 2 fs. The PfAQP

channel was divided into 0.25 A (1 A for urea) wide equidistant

sections parallel to the membrane with the center of each

section representing an umbrella center. Subsequently, the

solute molecules (glycerol or urea) were placed into the

channel at the umbrella center. To enhance sampling, several

solute molecules were placed in each pore keeping a distance

of 20 A between the solute molecules. Water molecules that

overlapped with the solute were removed. The central carbon

atom of glycerol or urea was restrained in z direction by a

harmonic umbrella potential (k=1000 kJ mol�1/nm2). In addi-

tion, the solute molecules were restrained into cylinders that

were centered along the respective channel. Accordingly, a

flat-bottom quadratic potential in the xy-plane was applied on

the restrained atom (radius rc = 6 A, kc = 400 kJ mol�1/nm2).

After energy minimization, each system was simulated for 2 ns

(4 ns for urea PMFs).

After removing the first 500 ps (1 ns for urea) for equilibra-

tion from each umbrella simulation, 1440 umbrella histograms

(360 for urea) were extracted from the z-coordinate of the

restrained atom. Subsequently, the umbrella positions were

corrected with respect to the center of mass of the corres-

ponding monomer. This procedure avoids a possible unphysical

flattening of the PMF due to fluctuations of the PfAQP

monomers within the tetramer. Visual inspection of the umbrella

histograms showed that multiple histograms overlapped at

each z coordinate. The PMF was calculated using a cyclic


implementation of the weighted histogram analysis method

(WHAM).38 The WHAM procedure incorporated the

integrated autocorrelation times (IACT) of the umbrella windows.

The IACTs were derived by fitting a double exponential to the

autocorrelation function of each window, allowing one to

analyically compute the IACTs. Because the IACT is subject

to large uncertainty in case of limited sampling (such as inside

the channel), we have subsequently smoothed the IACT along

the reaction coordinate using a moving average filter with a

width of 5 A.

Due to the cylindric flat-bottom restraint the umbrella

samplings yield a PMF which refers to a channel density of

one channel per cross section area of the cylinder. We also

corrected the PMF by a trapezoidal correction like the water

PMF above. In this case, Ac was chosen such that the entropy

of the solute in the flat-bottom quadratic potential equals the

entropy of a solute in a cylindrical well-potential of area Ac.

We found that this condition approximately holds if Ac is

computed via Ac = p(rc + 2sc)2 where sc = (kBT/kc)

1/2 is the

width of the Gaussian-shaped solute distribution at the edge of

the flat-bottom quadratic potential. Amono was estimated to

equal 10.7 nm2 and Ac was computed to 1.80 nm2, yielding a

correction of DGcyl = 4.4 kJ/mol. The statistical uncertainty of

the PMFs was estimated using bootstrap analysis as described

before,39 yielding an uncertainty of 2 kJ/mol (3 kJ/mol for

urea) at the main barrier and/or the main energy well.

3. Results

To quantify the water permeation through the different stu-

died aqua(glycero)porins, the permeability coefficient, pf, was

computed from equilibrium molecular dynamics simulations

(Fig. 2). The computed pf value of the wild type PfAQP is

3.3�0.2� 10�14 cm3/s. This value is nearly equal to the computed

value for hAQP1 and slighlty smaller than that of Glpf. In

addition, the mutation of Glu125 (at the C loop, Fig. 1) into

serine shows a reduction in the pf of 18% compared to the

wild type simulation. In contrast, the mutation of Arg196

(inside the narrow ar/R region of the pore, Fig. 1) into alanine

reveals a substantial increase (by a factor of two) in the water

permeability compared to the wild type simulation.

To identify the rate limiting regions in the pore that

determine the water permeation characteristics observed

above, the averaged pore radius and the PMF for water were

calculated (Fig. 3). In PfAQP, hAQP1 and GlpF, the pore

geometry remains essentially unchanged compared with the

hole profiles calculated from the X-ray structures,10 implying a

relatively rigid pore. In PfAQP (orange line) and GlpF

(cyan line) the pore profiles were found to be remarkably

similar, narrowing in the ar/R region down to 1.4 A radius,

and wider than the profile in hAQP1 (blue line). In addition,

the point mutation Glu125Ser (green line) does not modify the

pore geometry, whereas the Arg196Ala mutation (red line)

induces a widening of the pore to 1.7 A at the ar/R region.

The PMF for water permeation is also illustrated in Fig. 3.

In PfAQP (orange line), the PMF displays two major barriers

of 14.6 kJ/mol and 15.4 kJ/mol near the NPA region which is

not the most constricted region, and a third smaller barrier of

13.8 kJ/mol at the end of the ar/R region. In hAQP1 (blue line)

and GlpF (cyan line), the PMFs also have a main free energy

barrier at the NPA region, but they are smaller than the

barrier in PfAQP by 2.4 kJ/mol and 1.3 kJ/mol, respectively.

Furthermore, in PfAQP, the PMF displays eight local minima

inside the channel, and water molecules are predominantly

found at the positions of these minima. Consequently, the

effective water–water distance along the pore, computed as the

average distance between minima, is 2.6 � 0.3 A. At the same

region, the PMF for hAQP1 (GlpF) shows seven (eight)

minima separated by a water–water distance of 2.9 � 0.6 A

(2.8 � 0.6 A). In hAQP1, the water–water distance increment

is mainly observed between the NPA and the ar/R region.

Additionally, the Glu125Ser mutant (green line) does not

reveal significant changes in the PMF profile, whereas the

Arg196Ala mutant (red line) appears to have a complete

flattened profile at the ar/R region with a corresponding

decrease in the free energy barrier to a value less than

11 kJ/mol.

Finally, Fig. 4 depicts the PMFs for glycerol and urea,

derived from umbrella sampling simulations, for PfAQP

(orange line), and compared with previous molecular

dynamics studies for GlpF32 (cyan line). In PfAQP, the free

energy barrier at the ar/R region for glycerol is substantially

smaller than the existing for urea (approximately 21 kJ/mol

smaller). Moreover, for both solutes, glycerol and urea, the

free energy barrier at the ar/R region decreases in PfAQP

compared to that observed in GlpF. In addition, the PMF for

glycerol reveals new features in PfAQP that are not observed

in GlpF: first, there is a potential well at the intracellular

vestibule (z o �1.0 nm) instead of a free energy barrier.

Second, the potential wells at an intermediate region between

the intracellular vestibule and the NPA motifs are deeper in

PfAQP than in GlpF. Contrary to glycerol, the PMFs for urea

in PfAQP and GlpF were found to be similar between the

intracellular vestibule and the NPA motifs. Furthermore, for

both PfAQP and GlpF, glycerol crystallographic positions

(indicated with dots) agree favorably with the potential well

positions observed in the simulations.

Fig. 2 Single molecule water permeability coefficients derived from

equilibrium molecular dynamics simulations for the indicated proteins

and mutants.


4. Discussion

Here we present a computational study to characterize at the

molecular level the permeation of water, glycerol and urea

through the Plasmodium falciparum aquaglyceroporin

(PfAQP). Initially, the water permeation through PfAQP

was quantified by means of the single molecule osmotic

permeability coefficient, pf. Subsequently, the pore geometry

and the energetics for water, glycerol and urea transport

through PfAQP was analyzed by computing radius and poten-

tial of mean force profiles.

4.1 Quantifying the water transport activity

Let us first consider the absolute pf values predicted in our

simulations in light of permeation measurements and previous

computational studies. First, our computed values for the

reference (aquaporin) hAQP1 and (aquaglyceroporin) GlpF

(Fig. 2) are close to the experimental values, spanning between

4.6 � 10�14 cm3/s to 11.7 � 10�14 cm3/s for hAQP140–43 and

0.7 � 10�14 cm3/s for GlpF.44 Moreover, our predicted pf for

PfAQP is also close to these measurements. It should be noted

that there are no single-molecule pf measurements for PfAQP

reported so far to compare to directly. Second, our derived pfvalues are smaller than the values predicted in previous

computational studies on hAQP1 using the GROMOS force

field and the SPC water model31,45 by a factor of two.

Similarly, they are smaller than the values derived in studies

on hAQP1 and GlpF using the CHARMM force field and the

TIP3P water model46–48 at least by a factor of three. These

differences can be mainly attributed to the water diffusion

constant. For the SPCE water model (used in this study) the

diffusion constant is very close to the experimental value

(2.4 � 10�5 cm2/s). In contrast, for the SPC49 and the TIP3P33

water models (used in the mentioned previous computational

studies) the diffusion constant is overestimated by a factor of

1.5 and 2, respectively.

Let us now to consider the relative differences in the pf for

PfAQP compared to hAQP1 and GlpF. The computed pf for

PfAQP is close to the value predicted for hAQP1 (Fig. 2),

suggesting that PfAQP conducts water at a similar rates as

Fig. 3 Pore radius profiles (upper panels) and potential of mean force for water permeation profiles (lower panels) derived from equilibrium

molecular dynamics simulations. (a) Comparison between PfAQP (orange), hAQP1 (blue) and GlpF (cyan). (b) Effect of the point mutations

Glu125Ser (green) and Arg196Ala (red).

Fig. 4 PMFs for glycerol and urea permeation through PfAQP

(orange) compared with previous molecular dynamics studies for

GlpF32 (cyan). The dots correspond to the glycerol positions in the

PfAQP10 (orange dots) and Glpf13 (cyan dots) crystal structures.


hAQP1. This result is in excellent agreement with experimental

data from oocyte swelling assays,2 and supports therefore that

PfAQP is a highly efficient water channel. Interestingly, the pffor GlpF was observed to be higher than the values for PfAQP

and hAQP1, suggesting that GlpF also conducts water at

appreciable rates (comparable to hAQP1). A water permeability

for GlpF has been observed in previous computational

studies,31,46–48 experimental assays with proteoliposomes50

and reconstituded planar bilayers,44 but with the experiments

showing a lower permeation rate than the computational

studies. The reason for this discrepancy remains unknown.

A possible explanation would be simulation inaccuracies.

However, given the excellent agreement between the simula-

tion and the experiment for AQP1, and the consistent results

for GlpF in several different and independent simulations, this

appears unlikely. Another possible explanation could lie in

the problematic estimation of the reconstitution efficiency,

required to derive the copy number to estimate the experi-

mantal single-channel pf.

4.2 The energetics of water permeation

Further calculations of the pore geometry and the PMF for

water (Fig. 3) allowed us to identify the rate limiting regions

that determine the water permeation properties presented

above. The averaged radius profiles demonstrate that PfAQP

and GlpF have practically identical pore geometries, wide

enough to allow the passage of water and other solutes such

as glycerol and urea. Accordingly, the narrower pore observed

for hAQP1 constitutes one of the main factors for the exclu-

sion of large solute molecules in this aquaporin. The average

pore geometry from the simulations is remarkably similar to

that from the initial crystal structures10,13,14 (all compared in

ref. 10), indicating a rigid channel.

The pore geometry itself is not sufficient to explain the water

transport rates but the full energetics must be taken into

account. The major rate limiting region (highest barriers in

the PMF) for PfAQP is observed at the NPA region, located

exactly at the same place as for hAQP1 and GlpF (Fig. 3). In

all three cases, two rings of hydrophobic residues sit there

(Fig. 5), and they constitute the main barriers for water

passage. This is in perfect agreement with previous simulations

of hAQP1 and GlpF,31,51 and confirms that PfAQP has similar

water regulation mechanisms as other members of the family

of aquaglyceroporins. Furthermore, in PfAQP, the Leu192

residue may force the permeating water molecules to interact

more strongly with the Asn residue than in GlpF (where this

Leu192 is replaced by Met202, a less hydrophobic residue,

Fig. 5), leading to the observed higher free energy barrier,

for PfAQP as compared to GlpF. Surprisingly, hAQP1 has

smaller free energy barriers than PfAQP and GlpF in this critical

region, despite the fact that the protein-water interactions are

much stronger due to a narrower pore and the presence of the

larger Phe24 and Phe56 residues in this region (Fig. 5).31

Finally, from the free energy barriers, it could be expected

that PfAQP, GlpF and hAQP1 conduct water at rates in the

following order: PfAQP o GlpF o hAQP1. Our pf calcula-

tions predict the lowest rate for PfAQP. However, hAQP1 did

not show the highest rate but GlpF did. This apparent contra-

diction between the pf and the PMF for hAQP1 and GlpF may

be due to the fact that a permeation event contributing to the

pf (the translocation of one effective water molecule from one

aqueous medium to the other) implies the collective motion of

all molecules inside the channel,29,52 and therefore multiple

free energy barriers and not only the highest should be taken

into account when relating permeation rates to permeation

energetics.

Fig. 5 Top view of the two hydrophobic rings (upper and lower panels) located near the NPA motifs in the indicated proteins.


4.3 The effect of point mutations

We investigated the role of Glu125 and Arg196 on the water

permeation of PfAQP. On one hand, the Glu125Ser mutant

shows neither a substantial reduction in the pf nor an

increment in the free energy barriers compared to the wild

type simulation (Fig. 2 and 3(b)), contrary to what previous

mutation experiments demonstrated.9 The reduction in the

water permeability due to the Glu125Ser mutation was attributed

to the destabilization of the C loop and further disruption of

the hydrogen bonds between Arg196 and Trp124.9,10 How-

ever, the conformational change of the C loop may occur on a

longer time scale than the simulated time (100 ns), impeding to

be detected in our equilibrium simulations. In fact, the C loop

was observed to be rigidly anchored to the extracellular

vestibule (with an average backbone rmsd of 0.97 A with a

standard deviation of 0.26 A) in the wild type simulation, and

just slightly more flexible (with an average backbone rmsd of

1.06 A and a standard deviation of 0.36 A) in the Glu125Ser

simulation.

On the other hand, the Arg196Ala mutant reveals a widening

of the pore at the Ar/R region and a corresponding reduction

in the free energy barrier to water permeation, leading to a

significant increase in the water permeability (Fig. 2 and 3(b)).

This result confirms that Arg196 is a crucial residue for the

water conduction through PfAQP.

4.4 Permeation of other solutes

We studied the energetics of glycerol and urea conduction

(Fig. 4). Let us first analyze the free energy barriers at the ar/R

region for the different simulated solutes. Our simulations

predict that PfAQP conducts urea at a lower rate than glycerol

which is in excellent agreement with oocyte swelling assays.2 In

PfAQP, like in other aquaglyceroporins, glycerol molecules

arrange at the ar/R in such a way that its hydroxyl groups can

interact with Arg196, replacing water–Arg196 hydrogen

bonds, and its apolar backbone orients towards the hydro-

phobic face (Trp50 and Phe190 residues).10,13 In constrast,

urea molecules cannot find this compensatory effect (dictated

by the amphiphilic nature of the pore), because increasing

the carbonyl–oxygen interactions with Arg196 would lead to

unfavourable orientations of the amine groups facing the

hydrophobic residues, and therefore would increase the free

energy barrier as indeed observed in our simulations.

Focussing at the differences between PfAQP and GlpF, it is

intriguing to note that PfAQP has a reduced free energy

barrier for both glycerol and urea compared to GlpF at the

ar/R region. This suggests that PfAQP is a more efficient

glycerol and urea channel than GlpF, despite the fact that

both have identical residues in this critical region. A possible

explanation for this would be that fewer water–arginine

hydrogen bonds need to be replaced by the permeating solute

molecule in the case of PfAQP compared to GlpF, and there-

fore the energetic cost to replace water with a solute at that

position is reduced. Consequently, in PfAQP, the arginine

residue instead would form more additional hydrogen bonds

with the surrounding residues (namely, Ser200 and Trp124)

than in GlpF, as proposed by Newby et al.10 Indeed, the

histograms of the number of hydrogen bonds between the

solvent and the arginine residue computed in the equilibrium

simulations (Fig. 6(a)) show on average fewer hydrogen bonds

for PfAQP than for GlpF, and accordingly, more hydrogen

bonds between the protein and the arginine residue for PfAQP

than for Glpf (Fig. 6(b)).

In addition, the glycerol PMF for PfAQP reveals two

features not observed in GlpF: a potential well at the intra-

cellular vestibule and a putative binding site inside the channel

near the NPA region This binding site is expected to affect the

water permeability, particularly, at high glycerol concentra-

tions. It is therefore highly interesting to see if this glycerol-

dependent water permeability can be observed experimentally.

5. Conclusions

Here we present the first molecular dynamics study to charac-

terize at the molecular level the permeation of water and

other solutes through the Plasmodium falciparum malaria

aquaglyceroporin (PfAQP). Our simulations confirm that

PfAQP is a highly efficient water channel, that is able to

conduct water at single-molecule permeability rates comparable

to the hAQP1 and GlpF rates. Furthermore, we identified the

Fig. 6 Water–Arg196 (a) and protein–Arg196 (b) hydrogen bond

distribution in PfAQP (orange), hAQP1 (blue) and GlpF (cyan).


hydrophobic regions near the NPA motif as the main water

rate-limiting barriers, confirming therefore that PfAQP has a

similar water regulation mechanism as other members of the

family of aquaglyceroporins. Our simulations also support

that GlpF is a highly efficient water channel, with a water

permeability higher than that for hAQP1, as previously

observed in several different and independent simulation

studies.

We demonstrate that Arg196 located at the ar/R selectivity

filter plays a crucial role regulating the permeation of water

and other solutes such as glycerol or urea, as was previously

hypothesized from functional and crystallographic studies.

Our simulation results are consistent with a general permea-

tion mechanism for aquaglyceroporins in which water is rate-

limited by hydrophobic interactions in the NPA region. Other

solutes, such as glycerol or urea, must accommodate in the

ar/R selectivity filter, replacing water–arg196 hydrogen bonds

interactions and facing the hydrophobic Trp50 and Phe190

residues, a process that is favorable for glycerol but less

favorable for urea. In addition, in light of this mechanism,

our simulations suggest that PfAQP is able to conduct both

glycerol and urea at higher permeabilities than GlpF does,

because fewer water–Arg196 hydrogen bonds need to be

replaced by a permeating solute molecule in the case of PfAQP

compared to GlpF, a prediction to be further investigated and

validated in experimental studies.

The effect on the water permeability due to the mutation of

Glu125 (located at the C loop) to serine was found to be less

severe in our simulations than observed experimentally. This

could be attributed to the high stability of the C loop, rigidly

anchored to the extracellular vestibule, that would slow C loop

motions to time scales longer than the simulated time due to

the mentioned mutation.

This study is expected to guide further computational

and experimental studies in the search of putative blockers

of PfAQP, understanding of their mechanism of action, that

can hopefully be used to interrupt crucial physiological

processes of the malaria parasite such as the water regulation

and glycerol uptake.

Acknowledgements

We thank Erik Lindahl for providing us with the lipid

Berger-Amber parameters. We also thank Ulrike Gerischer

for carefully reading the manuscript. This work was supported

by grants from the European Commission, the Marie Curie

Research Training Network of Aqua(glycero)porins (MRTN-

CT-2006-035995), and by a Marie Curie Intra-European

Fellowship.

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